1Institute of Structural and Molecular Biology, University College London and Birkbeck College, University of London, London, UK; 2Department of Medicine,.
Downloaded from http://thorax.bmj.com/ on September 1, 2016 - Published by group.bmj.com
Spoken sessions association of premature emphysema in ZZ homozygotes who smoke.
S62
CHARACTERISATION OF A NOVEL “PSEUDO-Z” VARIANT OF a1-ANTITRYPSIN doi:10.1136/thx.2010.150938.13
1
M-P Nyon, 1L Segu, 2B Roussel, 3N Kalsheker, 2D A Lomas, 1K Thalassinos, B Gooptu. 1Institute of Structural and Molecular Biology, University College London and Birkbeck College, University of London, London, UK; 2Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK; 3 Division of Clinical Chemistry, University Hospital, Queens Medical Centre, Nottingham, UK 1
The Z (Glu342Lys) variant of a1-antitrypsin is common in populations of North European descent. The mutation causes individual a1-antitrypsin molecules to assemble into polymer chains in the endoplasmic reticulum of hepatocytes. Z homozygotes (PiZZ) have circulating levels of a1-antitrypsin w15% of normal and are predisposed to hepatic cirrhosis and severe, early-onset emphysema. The risk of clinically significant disease associated with the heterozygote PiMZ state is minimal. We describe a case phenotyped as PiZZ during family screening, but with surprisingly preserved circulating a1-antitrypsin levels. Genotyping revealed compound heterozygosity for the Z mutation and a novel, “pseudo-Z” mutation. Biochemical and ion-mobility mass spectrometry characterisation of pseudo-Z a1-antitrypsin showed that it readily populated a polymerogenic intermediate state under physiological conditions. Cell biological studies of a series of a1-antitrypsin variants indicated these effects involved disruption of a hydrogen bond stabilising the F-helix-linker region of the protein structure. These data strongly support the hypothesis that stability of this region co-regulates formation of the polymerogenic intermediate. Whilst the intermediate form of pseudo-Z a1-antitrypsin is more stable than that of the true Z variant, the resultant polymers all share a characteristic neoepitope. Pseudo-Z a1-antitrypsin is thus a useful model for in vitro screening of potential lead compounds to bind the polymerogenic intermediate state, improving the ability to develop novel therapies to treat a1-antitrypsin deficiency. Our data predict that the likelihood of severe disease in the PiZ/Pseudo-Z compound heterozygote state will be increased relative to the PiMZ state, but far lower than for PiZZ individuals.
complement previous structure-led approaches we have developed NMR spectroscopy and nanospray mass spectrometry as mediumthroughput screening tools for such ligands. The coupling of these techniques combines highly sensitive detection of ligand binding with assessment of binding sites, stoichiometry, cooperativity and binding constants. We have used the TTAI peptide, developed within an existing programme of drug design, as a test case. The data demonstrate highly co-operative, slow, tight binding of two copies of the peptide in adjacent parts of the a1-antitrypsin molecule. TTAI peptide binding is shown to induce widespread conformational change all over the molecule with the exception of b-sheet C. These data prove the utility of NMR spectroscopy and nanospray mass spectrometry in characterising ligand binding whilst providing a highly detailed template for use in specific screening for TTAI peptide-mimetic compounds.
S64
CHANGES IN PHYSIOLOGICAL PHENOTYPES OF a-1-ANTITRYPSIN DEFICIENCY WITH TIME doi:10.1136/thx.2010.150938.15
H Ward, M R Miller, R A Stockley. Department of Respiratory Medicine, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
Chronic Obstructive Pulmonary Disease (COPD), even due to a-1antitrypsin deficiency (A1AD), is recognised as having distinct radiological, physiological and clinical phenotypes. Little is known about disease progression in physiologically defined phenotypes.1 We have identified a subgroup of patients with a reduced FEV1 but normal gas transfer determined by the lower limit of normal (LLN), that is, with standardised residual (SR) value
